The use of fluorescence as a detection modality in biological assays is widespread and a diverse variety of procedures are available to generate fluorescence under assay conditions for detection by techniques such as fluorescence microscopy, fluorescence immunoassay and flow cytometry. Fluorescent signals may be generated using an enzyme to convert a non-fluorescent or substantially non-fluorescent substrate into a fluorescent product.
Such fluorescent enzyme substrates typically have two components that are coupled through a covalent linkage. One component is a fluorescent molecule that is capable of fluorescing by first accepting light energy and then emitting light energy. The other component is a masking group that prevents the fluorescent molecule from accepting or emitting light energy when the two components are covalently bound to one another, such that the molecule is non-fluorescent or substantially non-fluorescent. In the presence of an appropriate enzyme, cleavage of the covalent linkage takes place, thereby allowing the fluorescent molecule to absorb energy and emit fluorescence.
Tools exist for fluorescent imaging of cells in vivo using genetic reporters. Known reporters encode photoproteins that emit light that may be detected outside an animal's body by using optical cameras. With bioluminescence techniques, cells expressing a reporter gene product (e.g., luciferase) oxidize a substrate (e.g., D-luciferin), causing the substrate to emit light.
Optical imaging, which uses neither ionizing radiation nor radioactive materials, is emerging as a complement to nuclear imaging methods. The major limitation of light emitting probe is the high absorption and scattering that occur in biological tissues, which cause limited penetration of the light through the body. The currently available optical reporter genes emit light between about 400 nm and about 600 nm wavelengths.